1. Field of the Invention
The present invention relates generally to a method for automatic analysis of agglutination assays and more specifically, provides a fast, simple and automatic diagnostic system which does not require expensive equipment and enables registration of an agglutination assay and its result for later use. Furthermore, the system is suitable for screening and batch processing multiple samples.
2. Description of the Related Art
Agglutination reactions are valuable analytical tools that can be applied to many reaction systems in which multivalent binding between reactants is possible. A particle agglutination immunoassay is advantageous in that it requires only the mixing of a sample to be tested with a suspension of insoluble carrier particles (e.g., latex) sensitized with an antibody or an antigen. Typical examples are immunoassays which generally involve:
a) mixing a sample containing an antibody, with an antigen (corresponding to the antibody on the sample) and observing immunocomplex formation;
b) mixing a sample containing an antigen carrying at least two antigenic functions (bivalent or multivalent antigen) with the corresponding antibody and observing immunocomplex formation;
c) mixing monoclonal antibodies with a sample containing at least two different monovalent antigens and observing immunocomplex formation;
d) for any of the reactions mentioned above, coupling the antigen or antibody to particles, such as latex particles, colloids, etc.; and observing immunoagglutinate formation.
e) in the case of rapid plasma reagin (“RPR”) type reactions (e.g., for syphilis serodiagnostic), adding carbon as a visualizing agent and observing flocculation reactions;
f) for any of the reactions mentioned above, applying the above steps to antigens present on cell surfaces in which case the number of antigens per physical unit is normally a hundred or more, and in which case such cells may be agglutinated by monoclonal antibodies even if each antigen molecule is monovalent.
Such reactions are typically observed on the surface of a solid substrate such as a glass or plastic plate, or on the well of a microtitre plate. The solid surface is preferably colored to contrast with the color of the agglutinate.
The formation of visible agglutinates depends on the ratio of antigen/antibody. If a constant amount of antibody is mixed with increasing amounts of antigen three situations can be identified:
A pro-zone phenomenon, characterized by the presence of excess antibody in the test system and which is further characterized by non-occurrence of any visible phase reaction due to the inhibition of agglutinate formation by the excess antibody.
An equivalence zone characterized by the presence of antigen and antibody in optimum proportions and which is further characterized by enhanced agglutinateformation and visible phase reactions.
Post-zone Phenomenon characterized by the presence of excess antigen in the test system and which is further characterized by the nonoccurrence of visible reaction.
Pro-zone and Post-zone phenomena may be corrected by making serial dilutions of serum, thereby reducing the concentration of antigen or antibody in the test system, and optimizing the concentrations of antigen and antibody.
Agglutination reactions may also be performed with any set of molecules which bind to each other, provided that each of the reactants has at least two binding sites, or is coupled to a particle or otherwise linked together so that two or more binding sites per physical unit is created. Examples of systems other than antibodies/antigens that may form agglutinates are (poly)carbohydrates/lectins, biotin or biotinylated compounds/avidin or streptavidin, corresponding sequences of nucleic acids, any protein receptor and its corresponding ligand etc.
It can be appreciated that agglutination assays have been in use for years. Today, many agglutination assays are available to physicians for diagnosing various diseases, and an increasing number of such assays do not require that the patient's sample (e.g. blood, urine, saliva, stool) be sent to a diagnostic laboratory for analysis. Such in-office assays enable results to be obtained quickly and entered it to the patient's computer record. Test results can also be available for physicians in the emergency room.
Agglutination-based products for detection and quantification of analytes have been produced for a wide range of analytes. Very early on in the field, products were developed for the detection of human chorionic gonadotropic hormone (HCG) in urine, for the diagnosis of pregnancy. Two different principles were used: 1. products were made with antibodies on a particle surface, which gave agglutination in the presence of the analyte; and 2. products were made with antigen on the surface of the particles, and a reagent containing antibodies was added together with the test sample. In this second variant, agglutination took place in the absence or at low concentration of the analyte as a higher concentration of the analyte complexed with the antibodies and hindered the agglutination.
Furthermore, agglutination reagents for testing for drugs, including prescription drugs and most illegal drugs, and many non-proteinaceous hormones, such as testosterone, progesterone, oestriol, have been made.
The application of agglutination reactions is not confined to human or veterinary diagnostics, they function as great tools in other fields as well, including the agricultural industry, for detection of plant diseases (virus, bacteria and fungi) and industries which require monitoring of processes (e.g., the various food industries and the like).
It should be noted, however, that the examples given above are not considered to be a complete listing of the applications of agglutination assays and many other applications are possible.
Typical protein analytes for agglutination technology include C-reactive protein (CRP), transferrin, albumin, prealbumin, haptoglobin, immunoglobulin G, M, A and E, apolipoproteins, lipoproteins, ferritin, thyroid stimulation hormone (TSH) and other proteinaceous hormones, coagulation factors, plasminogen, plasmin, fibrinogen, fibrin split products, tissue plasminogen activator (TPA), betamicrogobulins, prostate-specific antigen (PSA), collagen, cancer markers (e.g. CEA andalphafoetoprotein), and several enzymes and markers for cell damage (e.g. myoglobin and troponin I and T).
Moreover, many agglutination test kits for infectious diseases have been made, including mononucleosis, streptococcus infection, staphylococcus infection, toxoplasma infection, trichomonas infection and syphilis.
Such reagents and reagent sets are either based upon detection of the infectious agent itself, or detection of antibodies produced by the body as a reaction to the infectious disease.
A typical medical technique for agglutination assay consists of mixing a sample with one or more agglutination reagents. Binding sites on the agglutination reagent(s) bond to corresponding sites on components of the sample, if present, and this binding results in agglutinates, which are visible clusters of bonded reagent and sample component. Thus, a desired reagent may be mixed with a sample and the presence of agglutinates in the mixture indicates the presence of the corresponding component in the sample. So a commercial latex particle agglutination test on slide it's used (like Toxocell Latex, made by Biokit of Barcelona, Spain).
A typical latex agglutination slide-based test, such as Toxocell Latex, made by Biokit of Barcelona, Spain is described below.
Primary Screening: Serum sample and latex reagent are mixed with a wooden stick on a slide section for approximately 5 minutes. Sample solvent is used as a negative control. After that, the slide is watched under direct and intense light for the presence or absence of agglutination. Results are recorded according to: positive reaction (3+ large aggregates on clear background, 2+ medium-sized aggregates on a slightly cloudy background, 1+ small aggregates on a cloudy background (for assay purposes, aggregates barely visible on milky background may be considered a positive indication) or negative reaction (absence of agglutination: milky uniform aspect).
Titration technique: the sample undergoes two serial dilutions with sample solvent over a single slide and is processed in accordance with the primary screening method described above. However, a titre of a given sample corresponds to the highest serum dilution that still presents a clearly visible agglutination (+1 according to the above scale). Provided that samples are tested in two fold dilutions, the real concentration will be in the range from that of the first and second dilution. With this technique, there is no way to determine the real concentration and in the technique is thus, useful in cases where it is the variation of concentration as opposed to the real concentration, that is of interest.
Although traditional agglutination reactions are, in fact, quantitative in nature, the interpretation of the result is traditionally qualitative.
However, since many of the analytes which may be the subject of such agglutination reactions are desired to be measured quantitatively, other and more complicated methods like ELISA, RIA, immunofiltration or immuno-chromatography methods have been used.
A few patents try to address the problem of quantitative vs. qualitative analysis in the context of agglutinate assays by the application of automatic procedure to a scanned image of the agglutinate assay. PCT International Application WO0005571, describes a device and method for the quantification of agglutination reactions based on a digital image of the agglutination reaction. Although this patent presents examples where samples with different concentrations produce different values in the measured features, no evidence is presented towards the reproducibility of such analysis. In some of the examples the obtained curves of measured features versus concentration have extremely small regions where quantification may be possible. Last but not least, the patent does not study the reproducibility of the process which is mandatory for automatic and general diagnostic systems. In several cases, agglutination patterns include great variations which are not accounted for when using these kinds of systems (see FIG. 2). The method proposed by PCT International Application WO0005571 does not address this problem. Since no preprocessing is applied to the image before feature extraction, great variations in the process can occur due to non uniform illumination, noise, dirt and bubbles, in addition to variations in the sample itself. Further, the procedure to obtain the reaction is completely manual, i.e., a wooden stick is used for the mixing of reagents and distribution thereof over a plate surface. This actually worsens the distribution of the agglutinates, as the particles will be formed erratically on an irreproducible fashion, causing the particles to increase in height. Accordingly, so any results obtained are typically inaccurate.
In U.S. Pat. No. 5,541,417, a similar method is proposed. A digital image of the agglutination reaction is obtained and processed. The quantification is based on a roughness index that captures the pixel local variations. These kinds of texture descriptors are efficient when dealing with uniform textures. However, as noted above, in the general case of agglutination reactions, great variations in the agglutination patterns are expected. As said before, these variations are often related with sample characteristics that can not be inferred a priori. Therefore, the roughness will produce different output values for samples with equal concentrations but different agglutination patterns. In a second embodiment the patent proposes to use a neural network to generalize local intensity variations. Neural networks must be trained using a sufficiently rich training data. This is a limitation in diagnostic systems where reagents may change over time, producing variations in the agglutination patterns. Therefore, every time changes are made to the system the network must be trained and sent to all the users of the system. On the other hand, as mentioned above, agglutinations with similar concentrations may have different agglutination patterns (see FIG. 2) and a neural network based only on a roughness measure will have problems to accurately quantify the sample.
Another example of the application of this technique involves blood group serology, where agglutination is the result of mixing red cells (containing a particular antigen) with a serum containing the corresponding antibody. Blood typing tests are done before a person receives a blood transfusion and to check a pregnant woman's blood type. Human blood is classified, or typed, according to the presence or absence of certain markers (called antigens) on the surface of red blood cells. The most important antigens are blood group antigens (ABO) and the Rh antigen. Therefore, the two most common blood typing tests are the ABO and Rh tests.
With respect to this type of agglutination assay, PCT International Application WO8907255 attempts to solve a normal operation problem with the automatic detectors for hemoagglutination test. In the normal procedure, the way in which the agglutinate deposits over the walls and bottom of the sample cell (or plate depression), make the automatic detection of the agglutination difficult. Normally a small button is formed on the centre of the reaction surface, and other deposits are scattered unevenly across the surface. The method appears to solve those problems only in cases when a positive/negative detection is needed, comparing the absorbance values of the centre and periphery zones of the bottom of the cell (or plate). However the described method cannot be applied to diluted samples, usually found when the analyte concentration is low, and/or the absorbance difference between both zones is low and each absorbance value is close to zero (because the high dispersion of the agglutinates).
Thus, traditional agglutination assays have been carried out only semi quantitatively, and the interpretation of results obtained therefrom are subject to human error inaccuracy and are often not reproducible.